Discovery of a Thrombin-Targeting Anticoagulant Peptide From Whitmania pigra via a “Computation-Guided Experimentation” Strategy

Targeting thrombin to screen safe thrombin inhibitors from natural plants and animals is a critical direction in anticoagulant drug development. This study aimed to screen thrombin inhibitors from the nonbloodsucking leech Whitmania pigra (WP) and elucidate the mechanism of anticoagulation through a “computation-guided experimentation” strategy. A peptide library was constructed from WP hydrolysates, and virtual screening was performed using molecular docking and dynamics simulations. A novel thrombin-targeting anticoagulant peptide PEP^WP (LRELEDALEQER) was screened out from the peptide library and validated through in vitro/in vivo experiments. PEP^WP significantly prolonged thrombin time (TT) and prothrombin time (PT) in a dose-dependent manner in vitro, indicating its role in the common and extrinsic coagulation pathways. Surface plasmon resonance (SPR) analysis then confirmed strong thrombin binding (K_d = 7.242 × 10⁻⁶ mol/L). Furthermore, PEP^WP prolonged TT while reducing blood viscosity in acute blood stasis rats. Finally, structural analysis revealed that PEP^WP bound to Exosite II of thrombin. Arg233 and Arg101 were the key residues for the binding. In conclusion, PEP^WP exhibited good anticoagulant activity and significant application potential.